Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

Abstract

BackgroundProteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown.MethodsWe produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches.ResultsWe show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell.ConclusionsWith the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.

Prion protein glycans reduce intracerebral fibril formation and spongiosis in prion disease

“…First, using 4 conformationally distinct prion strains, we show that unglycosylated PrP favors extracellular plaque formation and induces severe spongiform degeneration in the brain. Second, using an antibody specific for shed PrP (38), we show that the plaques are highly enriched in ADAM10-cleaved PrP. Third, using mass spectrometry and immunohistochemical labelling, we show that these unglycosylated, ADAM10-cleaved prions tightly bind heparan sulfate (HS) and HS colocalizes to plaques.…”

Domain-specific quantification of prion protein in cerebrospinal fluid by targeted mass spectrometry

“…This may account for the much lower response of this peptide in CSF vs. the 15 N standard (Figure 2). For the C-terminal peptide ESQAYYQR, our assay might not detect proteolytically shed PrP if the cut site for ADAM10, the predominant PrP sheddase 45 , in human PrP is homologous to its reported cut site in rodent PrP 8,46 . For the most N-terminal peptide monitored, RPKPGGWNTGGSR, the presence of a retained N-terminal methionine three residues upstream of this sequence in bacterially expressed PrP, detected here ( Figure S3) consistent with reported N-terminal methionine excision patterns in E. coli 47 , could alter its trypsin digest efficiency relative to brain and CSF PrP.…”

“…PrP is an extracellular GPI-anchored protein that can be shed from the plasma membrane by ADAM10 and other peptidases 7,8 . CSF PrP is predominantly soluble and full-length 9 , suggesting that it originates chiefly from this proteolytic shedding near the C terminus, although lower molecular weight fragments of PrP have also been identified in CSF 10 , which may originate from other endoproteolytic events 7,11 , and anchored PrP is also released from cells on exosomes 12 .…”

Prion protein—Semisynthetic prion protein (PrP) variants with posttranslational modifications

“…Already 10 years ago, it was suspected that reports on the function of PrP represent just specific aspects of a more complex physiological role of PrP C . Causes for the functional diversity of PrP C might not only be its alternating transient binding partners in different cellular locations but also its proteolytic processing . For once, PrP fragmentation may inhibit association with some binding partners while possibly allowing new interactions with others.…”

“…23 Causes for the functional diversity of PrP C might not only be its alternating transient binding partners in different cellular locations but also its proteolytic processing. 124,125 For once, PrP fragmentation may inhibit association with some binding partners while possibly allowing new interactions with others. Then again, the cleaved products may act as soluble ligands facilitating protein interactions over large distances.…”